To address the problem of bacterial contamination, several test methodologies have been developed. The classical methods are based on cultivation of bacteria on a nutrient media supporting growth. After approximately 2-14 days, bacteria capable of growing on solid medium have multiplied to a level where colonies become visible and can be counted, and bacteria capable of growing in fluid medium can be measured by e.g. optical density or dry weight. Efforts have been made to expedite and simplify the detection process. Among these efforts have been methods based on measurements of radiometry, impedance, chemiluminiscence and fluorescence.
Radiometric approaches for identifying bacterial contamination generally utilize incorporation of a radioactive nutrient by the bacteria. The radiolabelled bacteria can be isolated and quantified by following the radiolabel. This methodology has several undesirable drawbacks. Although very sensitive, it utilizes radioisotopes which can be expensive and difficult to handle.
Methods based on electrical impedance typically include a cultivation step. As the microorganisms grow, changes in impedance of the nutrient medium can be detected and correlated to the microbial growth. Methods based on electrical impedance, although more rapid than classical cultivation, are still slow, involving an incubation period of 1-4 days.
ATP is detected by chemiluminiscence. Detection and/or quantification of bacteria by use of detection of ATP is rapid and can be performed within minutes. However, ATP is ubiquitous and the kinetics of ATP-derived luminescence is complex, qualities that lowers the robustness of methods based on this principle. Furthermore, the turnover of ATP in the cells is very rapid and the ATP content of cells may experience huge variations in a short time period e.g. when cells goes from growth to starvation.
Several methods have been described in the prior art based on the enzymatic degradation of a fluorescently labelled umbelliferone substrate with concomitant monitoring of the fluorescence derived from the released umbelliferone. Detection or quantification of bacteria by use of enzyme activity may also be susceptible to interference from non-bacterial sources although this interference appears less significant. Furthermore, the amount of product (fluorescence) formed per time unit is linear. Minimized interference and simple kinetics render measurements of bacteria by use of enzyme activity more robust.
U.S. Pat. No. 4,591,554 (Koumura et al.) discloses a method for rapidly detecting microorganisms utilizing nonfluorescent umbelliferone derivatives such as 4-methyl-umbelliferyl-β-D-galactoside, 4-methyl umbelliferyl-α-D-galactoside, 4-methyl umbelliferyl-phosphate, and 4-methyl umbelliferyl-pyrophosphate. Fluorescence of the liberated umbelliferone moiety is induced at 360 nm and monitored at 450 nm. Enhancement of sensitivity is obtained through a cultivation step for 1-12 hours.
U.S. Pat. No. 5,518,894 (Berg) discloses a rapid method to detect the presence of coliform bacteria. This method comprises a concentration step (filtration) in combination with a cultivation step to increase the number of target bacteria present. The fluorescence of hydrolysed umbelliferone derivative is monitored as an indication of the presence of coliform bacteria.
U.S. Pat. No. 5,610,029 (Ehrenfeld et al.) discloses a culture medium for the detection of presence or absence of target microorganisms in a sample. This culture medium includes various nutrients and growth factors, as well as a fluorescent metabolite (4-methyl umbelliferyl-β-D-glucuronide).
All the above mentioned methods based on detection of fluorogenic detection of enzyme activity, utilises a cultivation step which typically leads to a total performance time of 6-72 hours, which in many cases do not satisfy the demands for performance of a rapid method, let alone a method which is performed in situ.
U.S. Pat. No. 5,089,395 (Snyder et al.) discloses use of a nonfluorescent umbelliferone derivative which is enzymatically converted to a fluorescent product to detect the presence of bacteria. In this method there is no cultivation or concentration step. Due to the lack of these steps, the method is not highly sensitive and needs a high concentration of bacteria of a least 1000 /ml and typically higher concentration are acquired.
U.S. Pat. No. 5,968,762 (Jadamec et al.) discloses a method that uses a nonfluorescent umbelliferone derivative which is enzymatically converted to a fluorescent product to detect the presence of bacteria. The invention relates to measuring the fluorescent intensity ratio of the metabolised fluorescent product at a specific wavelength to the metabolizable fluorescent conjugate at a second specific wavelength. A detection time of approx. 80 min for detecting a concentration of 310 (cfu/ml) is given (cfu=colony forming unit).
Membrane filtration of liquid samples is commonly used for investigating liquid samples for bacteria. The sterile membrane filter is placed in a closed device which can be sterilized and the bacteria are collected on the filter. The filter can then be placed on an agar-containing nutrient medium where the colonies can be enumerated following a cultivation process. The filter may also be treated with a fluorogenic dye which is incorporated into the bacteria which then can be enumerated by laser induced fluorescence. All microbiologists who use membrane filtration are familiar with the care that needs to be taken in order to secure a sterile handling the filters. When detecting small numbers of bacteria a filtration step may easily introduce pollutions rendering the process unreliable and highly dependent on operator skill.
Accordingly, what is needed in the art is a rapid method to detect the presence of bacteria in a sample that is simple to perform, robust and reliable.